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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Analytical Support: 21 September 2017 and 10 June 2018, Definitive Assay: 06 -08 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted April 13 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the start of the test (0 hours) and at media renewal at 24 hours, 10 mL samples of freshly prepared test media were taken from the control and the test media preparation vessel for chemical analysis.
At 24 and 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media from the test and control groups.
Vehicle:
yes
Details on test solutions:
The dilution water used in the study was Elendt M4 medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile Daphnia magna (Straus), less than 24 hours old, were taken from healthy parental laboratory cultures, held and maintained at the test facility. The culture was originally initiated from ephippia obtained from MicroBio Tests Inc., Belgium.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
19.2-19.5 ºC
pH:
7.5-7.8
Dissolved oxygen:
93-100 % saturation
Nominal and measured concentrations:
Nominal Measured concentration in mg/L

0 h 24 h (old media) 24 h (new media) 48 h (old media)

100 mg/L 111 76.2 110 92.5
Details on test conditions:
Range-finding Test
The range-finding test was conducted at nominal concentrations of 0.10, 1.0, 10 and 100 mg/L under static test conditions. A control group was also included. Duplicate test vessels were prepared for the control and test groups. Chemical analysis of the test preparations was not conducted during the range-finding test.

Solubility/Stability Trial
Following completion of the range-finding test, initial trials were conducted to determine the solubility and stability of the test substance in test media. Initial trials showed highly variable analytical results.

During analytical method development it was realised that the test substance was highly unstable in the test media. Further work indicated that the DT50 was <1 hour. As recommended by the OECD Series on Testing and Assessment, No. 23 (2000); (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures), the testing was therefore conducted on the degradation product 3-hydroxy-4-oxo-3,4-dihydronaphthalene-1-sulfonic acid.

A solubility/stability trial was therefore conducted to determine the solubility and stability of the degradation product in test media over the duration of the test.
Test concentrations of 100 mg/L were prepared in triplicate in Elendt M4 medium. The preparations were left under laboratory conditions for ca 2 hours to fully degrade. Samples were taken for analysis from each preparation at 0 hours (following degradation). The preparations were then left under test conditions for 48 hours after which further samples were taken for chemical analysis.

Definitive Test
Based on the results of the range-finding, for which only the key findings have been reported, the definitive test was conducted as a Limit Test at a single nominal concentration of 100 mg/L. A control group was also included.
The test was conducted using semi-static test conditions with renewal of test media at 24 hours.
The test vessels (nominal volume 100 mL) were glass tall form beakers covered with a clear lid. Four replicate test vessels were prepared for the control and each test concentration containing 50 mL of the appropriate media.
At the start of the test and media renewal at 24 hours, ca 50 mg of test substance was dissolved in 500 mL of Elendt M4 medium. The preparation was left at room temperature for approximately 2 hours to degrade prior to adding to the test vessels. A control group was prepared by adding Elendt M4 medium only to the control vessels.
Five juvenile Daphnia magna, less than 24 hours old, were added to each test vessel, using a wide bore pipette to avoid damaging the animals during transfer. After 24 hours, the daphnids were transferred to fresh media. The Daphnia magna were not fed during the test.
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

Water Quality and Environmental Conditions
The test was conducted in a light and temperature controlled laboratory with a
16-hour light: 8-hour dark photoperiod.
The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined in freshly prepared test media at the start of the test and at media renewal at 24 hours and in the old media at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
At the end of each exposure period the test water quality measurements were conducted using pooled replicate samples of old test media.

Chemical Analysis
Concentrations of 6-hydroxy-5-oxo-5,6-dihydronaphthalene-1-sulfonic acid were determined by diluting aqueous samples with Elendt medium as required to bring the response within the calibration range. Samples were analysed by injection onto a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) system.
At the start of the test (0 hours) and at media renewal at 24 hours, 10 mL samples of freshly prepared test media were taken from the control and the test media preparation vessel for chemical analysis.
At 24 and 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media from the test and control groups.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: degradation product
Basis for effect:
mobility
Remarks on result:
other: degradation product
Details on results:
The numbers of immobilised Daphnia magna at 24 and 48 hours, following exposure are presented below. The percentage immobility data is summarised below.

----------------------------------------------------------------------------------------------------------------------------------------------------------------
Time-weighted Number of Daphnia magna % %
mean measured concentration (mg/L) exposed Immobility at 24 hours Immobility at 48 hours
----------------------------------------------------------------------------------------------------------------------------------------------------------------
Control 20 0 0
97 20 0 0
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Toxicity values and the corresponding NOEC and LOEC values are presented in the following table. Values are presented in terms of time-weighted mean measured concentrations.

Time-weighted mean measured concentration (mg/L)
24-hour 48-hour
EC50 >97 >97
LOEC >97 >97
NOEC 97 97

The 48-hour EC50 value was calculated to be >97 mg/L. The corresponding NOEC was considered to be 97 mg/L.
Results with reference substance (positive control):
EC50 (48h) = 1.366 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 value was calculated to be >97 mg/L. The corresponding NOEC was considered to be 97 mg/L.

Description of key information

The 48 -h EC50 value was determined to be greater than 97 mg/L.

Key value for chemical safety assessment

Additional information